1. Field of the Invention
This invention relates to a method for producing the stable transformants of coffee plants.
2. Description of Related Art
Coffee is a commercially important woody shrub planted in a large scale for harvesting its beans. Among more than 80 species, the most economically important are Coffee arabica (2n-44) and C. canephora (2n-22). In C. arabica, genetic diversity is limited by conventional breeding because of its self-pollination characteristic, and the plants are highly sensitive to pests and diseases. C. canephora, used for instant coffee powder products, is a cross-pollinated specie but has low production quality. Conventional breeding of coffee is difficult because of the long duration of cultivation to set seeds. Molecular breeding, therefore, is a desirable technique for the genetic improvement of coffee species, although production of transgenic coffee plants via gene transformation has generally been considered problematic.
Plant regeneration via in vitro tissue culture is a basic system for achieving genetic transformation, and there have been many reports involving somatic embryogenesis in coffee plants (Staritsky, 1970; Hatanaka et al. 1991; Menxc3xa9ndez-Yuffxc3xa1 and Garcxc3xada, 1996). However, data for genetic transformation of coffee are limited. Barton et al. (1991) obtained transformants from electroporated protoplasts of C. arabica, but the cultured protoplasts did not develop into whole plants. Spiral et al. (1993) reported the transformation of coffee (C. canephora) by co-cultivation of Agrobacterium rhizogenes with microcut-somatic embryos. However, the efficiency of transformation was very low. Van Boxtel et al. (1995) reported only transient expression of GUS genes on the surfaces of coffee leaf tissues following biolistic delivery.
Agrobacterium tumefaciens-mediated transformation is considered to be best for plant transformation because of the availability of vectors. Despite such advantage, no report has been presented of successful coffee transformation using Agrobacterium tumefaciens strains, except for GUS positive transgenic callus induction at a low frequency reported from Ocampo and Manzanera (1991).
This invention provides the successful genetic transformation of Coffea canephora using Agrobacterium tumefaciens EHA101 harboring pIG121-Hm from embryogenic calli.
Embryogenic calli were induced from leaf explants of Coffea canephora on McCown""s woody plant medium (WPM) supplemented with 5 xcexcM N6-[2-isopentenyl]-adenosine (2-iP). These calli were co-cultured with Agrobacterium tumefaciens EHA101 harboring pIG121-Hm, containing xcex2-glucuronidase (GUS)-, hygromycin phosphotransferase (HPT)- and neomycin phosphotransferase II (NPT II) genes. Selection of putative transgenic callus was performed by gradual increase in hygromycin concentrations (5, 50, 100 mg/l). The embryogenic calli surviving on a medium containing 100 mg/l hygromycin showed a strong GUS positive reaction with X-gluc solution. Somatic embryos were formed and germinated from these putative transgenic calli on WPM medium with 5 xcexcM 2-iP. Regenerated small plantlets with shoots and roots were transferred to a medium containing both 100 mg/l hygromycin and 100 mg/l kanamycin for final selection of transgenic plants. The selected plantlets exhibited strong GUS activity in leaves and roots as indicated by a deep blue color. GUS and HPT genes were confirmed to be stably integrated into the genome of the coffee plants by the polymerase chain reaction (PCR).
Moreover, the inventors have succeeded in production of a transgenic plant of Coffea arabica, wherein phosphinothricin acetyl transferase (BAR) gene was incorporated to render resistance against herbicide. Commercially, Coffea arabica is more valuable than Coffea canephora. Embryogenic calli derived from Coffea arabica were induced from leaf explants of coffee on Murashige and Skoog (MS) medium supplemented with 10 xcexcM N6-[2-isopentenyl]-adenosine (2-iP). These calli were co-cultured with Agrobacterium tumefaciens EHA101 harboring pSMBuba, containing herbicide resistant BAR gene and hygromycin phosphotransferase (HPT) gene. Selection of putative transgenic callus was performed by gradual increase in hygromycin concentrations (25, 50 mg/l). The embryogenic calli maintained on MS medium with 50 mg/l hygromycin and 10 xcexcM 2-iP. Prolonged culture of embryogenic callus induced somatic embryos. Germination of somatic embryos strongly enhanced by GA3 treatment and developed into transgenic plantlets after 2 months of culture. Transgenic embryogenic callus, somatic embryos and small plantlets were tolerant to 2 mg/l Bialaphos. Whereas non-transformed ones were dead after 1 month. Prescence of HPT and BAR genes in those transgenic plantlets was confirmed by the genomic PCR and Northern assays.
This invention provides a method to incorporate an exogenous gene using Agrobacterium tumefaciens mediated method. Embryogenic calli were induced from leaf explants of coffee plants. The embryogenic calli thus obtained were infected by Agrobacterium tumefaciens, harboring a plasmid containing an exogenous gene to be incorporated and hygromycin phosphotransferase (HPT) gene. Putative transformed calli were selected using the hygromycin resistance as an indicator. And then somatic embryos were induced from the putative transformed calli. Transformed plantlets can be regenerated from the somatic embryos thus obtained.
Various species of coffee plants can be transformed using the method of this invention. The coffee plant species may preferably be cultivative coffee species such as Coffea arabica, Coffea canephora, Coffea liberica and Coffea dewevrei. 
Theoretically, any exogenous gene can be incorporated into coffee plants by the method of this invention. The exogenous genes to be incorporated may preferably be caffeine synthetase gene, herbicide resistance gene such as phosphinothricin acetyl transferase (BAR) gene, insect injury resistance gene such as Bacillus thuringiensis gene, and disease resistance gene such as chitinase gene and glucanase gene.
Other and further objects, features and advantages of the invention will appear more fully from the following descriptions. It is to be understood that, examples mentioned above and description of detailed embodiments are not to be intended to limit the range of this invention.